Neurogenetics of Reward Sensitivity We have been studying how spontaneous genetic variation that influences reward sensitivity, and which could be adaptive in certain contexts, may also increase risk for alcohol related problems. In both humans and rhesus macaques, there are polymorphisms in the mu-opioid receptor gene (OPRM1) that influence affinity of the receptor for its endogenous ligand, -Endorphin. We and others have shown that these polymorphisms predict increased alcohol-induced euphoria, suggesting them to have gain-of-function roles. Other, unrelated studies support this, in that HPA axis activity (which is restrained by endogenous opioids through this receptor) is decreased among human carriers of the alternative allele. We find this to be true in macaques as well and, moreover, find that this effect applies to a wide variety of stressors and extends across life history stages (Schwandt et al, 2011). We also have demonstrated it to relate to individual differences in response to natural rewards, as shown by measures of both reward bias in humans (Lee et al, in press) and social attachment in both human and macaque (Barr et al, 2008;Higham et al, 2011;Copeland et al, 2011). Another receptor in this family whose activity may influence reward system processing is the nociceptin receptor (OPRL1). Humans, rhesus macaques, and dogs all have missense variants at the c-terminal end of the receptor. These are present at high frequency, despite the fact that the region is under intense selection (high interspecific conservation and low intraspecific variation in dog and mouse). Whether variation at this gene influences alcohol consumption or other reward-dependent behaviors is currently under examination. Using Convergent Findings Across Species to Identify New Candidate Genes for Alcoholism Risk A variety of animal species have been used to model certain aspects of alcohol use and addiction. Strains of rats that have been selectively bred for alcohol preference have been enormously informative for identifying relevant intermediate phenotypes and for determining genetic contributions to alcohol consumption and related traits, and studies performed in macaques have repeatedly demonstrated their value for identifying genotype-phenotype relationships that translate to the human condition. In collaboration with LNG, we have performed a study that combines varied analytical approaches across species in order to home in on genes that may contribute to risk for alcohol use disorders in humans. RNA-Seq was performed in brain for two selectively bred lines of rats (P and NP) to identify differentially expressed genes. Among the 485 differentially expressed (FDR <0.05) genes for the P and NP rat lines, the Fam111a gene showed an over twenty-fold difference between the P and NP lines. We then performed deep sequencing of the FAM111A gene in rhesus macaque, and identified 13 polymorphisms (SNPs), two of which were nonsynonymous and one of which was present in the proximal promoter. The promoter SNP was predicted to disrupt a number of important regulatory sites. The nonsynonymous SNPs did not significantly affect alcohol consumption, but animals carrying the promoter SNP exhibited alcohol consumption levels that were 2.5 times higher than those observed in animals homozygous for the ancestral allele. The FAM111a gene encodes a serine-endopeptidase, but little is known about its exact function. It has been shown that rats bred for a model of depression exhibit increased expression levels for FAM111A RNA in brain compared to resistant lines, and studies in humans show links between peripheral FAM111A expression levels and nicotine dependence. These data suggest that the FAM111A gene may contribute to risk for affective and addictive disorders in humans. Genomic and Epigenomic Studies Performed Using Next-Generation Sequencing In 2009, a high-profile meta-analysis was performed that called into the question the validity of many of the G x E findings that had been reported in the human literature. Debates surrounding this issue are ongoing, calling out for studies that might verify the validity of G x E interaction results by elucidating mechanisms by which they might occur. One mechanism by which stress could interact with genotype is via epigenetic modifications. The emergence of next generation sequencing technologies has broadened our potential for discovery of epigenetic effects. We have used these approaches and have been performing both ChIP- and MDP-Seq in order to examine effects of early stress on histone and DNA modifications in tissue derived from stress-sensitive brain regions (ie, hippocampus). Among the genes shown to be epigenetically regulated by stress in macaque were SLC6A4 (serotonin transporter), BDNF, CRH/CRHBP, NPY, and the glucocorticoid and oxytocin receptor genes (NR3C1 and OXTR), to name a few. The serotonin transporter finding was of great interest, since functional polymorphisms in the regulatory region for this gene have been shown to interact with early adversity in multiple primate species to predict vulnerability to stress-related problems (Barr, 2011;Schneider et al, 2011;Schwandt et al, 2011). The most robust result from this study was that obtained for the oxytocin receptor gene, OXTR. Results for OXTR held with four different analytical approaches, demonstrating stress-induced decreases in levels of both H3K4me3 binding and RNA expression levels in hippocampus. In order to provide functional validation for a role of epigenetic regulation at OXTR in G x E interactions, we then performed analyses to determine whether a non-synonymous SNP in the ligand-binding domain of OXTR would interact with early stress to influence relevant behavioral phenotypes. We found that in animals reared under conditions of early adversity, this genotype predicted increased levels of anxiety-like behavior and alcohol consumption. A larger sample is currently being genotyped in order to verify these effects. Oxytocin is a neuropeptide that produces affiliative, amnesic and anxiolytic affects. Given its roles in some of these processes, it has been proposed as a potential therapeutic agent for the treatment of anxiety and stress-related disorders, including PTSD. Functional variation that influences oxytocin system function may, therefore, be a particularly good candidate for performing gene x stress interaction studies in humans.